Copper is an essential nutrient and is required by a number of distinct cellular processes. It is important as a co-factor in anti-oxidant defense, in neurotransmitter biosynthesis and a number of other important biochemical transformations. Although the presence of low cellular levels is essential for human health, elevated levels are toxic. Thus, copper entry and elimination from cells and tissues are highly important pathways. Recently the protein responsible for mediating the entry of copper into human cells has been identified and is called hCtr1. This integral membrane protein consists of 190 arninoacid residues and appears (from hydropathy analysis), like its yeast homologues to span the membrane three times. It has been demonstrated to be essential for normal embryonic develpment. The PI of this application has recently achieved the successful heterologous expression of hCtr1 in insect cells infected with baculovirus particles containing the eDNA for hCtr1. The present application aims to provide the first detailed characterization of structure-function relations in hCtr1 and will investigate the mechanism of copper transport mediated by hCtr1. The studies will combine functional assays of isotopic copper uptake with mutagenic analysis of novel hCtr1 molecules. These studies will be augmented by an examination of trafficking of endogenous levels of hCtr1 in mammalian cells using confocal microscopy and specific Ab's produced by the PI's laboratory. The results of these studies will provide an essential starting point for an understanding of an important aspect of copper regulation in humans. Disruptions in copper metabolism and homeostasis have been associated with several neurodegenerative conditions and the development of new therapeutic strategies requires greater knowledge of the regulatory pathways in normal and diseased states.